Peripheral arterial disease (PAD) is a major complication of diabetes and is a leading cause of amputations of digits or limbs. My laboratory has focused for the past 15 years on the molecular regulation of genes in hypoxic stress. In the previous cycle, three major discoveries linked RAGE to regulation of acute responses to hypoxia: First, in cultured endothelial cells and monocytes/macrophages, acute exposure to hypoxia resulted in rapid release of AGE-reactive epitopes within minutes of hypoxic stress. Second, in the intact heart and in primary murine aortic and human aoilic endothelial cells, RAGE regulated rapid upregulation of Egr-1 in a manner suppressed by deletion or knockdown of RAGE and by aminoguanidine, an AGE inhibitor. Third, in collaboration with Project 1, we showed that monocytes/macrophages devoid of RAGE orthe RAGE cytoplasmic domain binding partner, diaphanous-1 or mDia-1, did not upregulate Egr-1 in acute hypoxia. To address the diabetes-relevant implications of these findings, we employed an in vivo model of hind limb ischemia induced by unilateral femoral artery ligation. Diabetes significantly impairs vascular repair mechanisms in this model. Our preliminary data reveal a fascinating and unexpected switch in RAGE signaling in acute vs. chronic oxygen deprivation ... whereas RAGE signaling upregulates Egr-1 in acute hypoxia, in hind limb ischemia, particularly in diabetes, RAGE suppresses adaptive induction of Egr-1 and, in parallel, reduces angiogenesis and blood flow recovery. In this application, we will probe the effects of specific deletion of RAGE in either monocytes/macrophages or endothelial cells to dissect the mechanisms by which RAGE action suppresses angiogenesis and blood flow recovery. Together with Projects 1&3, we will discern the fine-tuning mechanisms by which RAGE acts in common cardiovascular stresses, as all three Projects will use Affymetrix arrays to develop an integrated picture of RAGE signaling in distinct cell types and situations. We will use all three Cores in all five years ofthe Program.